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Photoelectric sensor for detection of kinase activity on the basis of local area surface plasma resonance

A plasmon resonance, photoelectric sensor technology, applied in instruments, measuring devices, scientific instruments, etc., can solve the problems of inability to directly apply biomolecular detection, influence of biomolecular activity, and low amount of enzyme immobilized in glassy carbon electrodes, and achieve Enhanced localized surface plasmon resonance effect, ultrasensitive detection of kinase activity, and improved photoelectric conversion efficiency

Active Publication Date: 2015-11-04
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to TiO 2 / ZnO is a wide bandgap oxide, which requires high-energy ultraviolet light to be excited, and ultraviolet light has an impact on the activity of biomolecules, so it cannot be directly applied to the detection of biomolecules
[0004] However, the working electrodes in existing biosensors are mainly glassy carbon electrodes or gold electrodes. Among them, glassy carbon electrodes cannot be directly immobilized with enzymes by physical or chemical methods, and can only be treated by other treatments on the surface of glassy carbon electrodes, such as Chemical cross-linking or sol-gel allows a small amount of enzymes to be immobilized on the surface of glassy carbon electrodes
Due to the low amount of immobilized enzymes in the glassy carbon electrode, its sensitivity has not reached the ideal level
However, gold electrodes usually only immobilize enzymes containing or modified with specific groups such as sulfhydryl groups, which has certain limitations.
In addition, glassy carbon electrodes and gold electrodes must be polished and activated before use, which is a cumbersome process

Method used

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  • Photoelectric sensor for detection of kinase activity on the basis of local area surface plasma resonance
  • Photoelectric sensor for detection of kinase activity on the basis of local area surface plasma resonance
  • Photoelectric sensor for detection of kinase activity on the basis of local area surface plasma resonance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Fabrication of Photosensors

[0044] 1. Preparation of probes:

[0045] The synthesis of nano gold: 100ml 0.01% (w / v) chloroauric acid aqueous solution is placed on magnetic stirrer and is heated to boiling, after that 0.5mL 1% (w / v) sodium citrate aqueous solution is added rapidly to boiling chloroauric acid In the solution, when the solution turns wine red, the heating is stopped to obtain gold nanoparticles, which are stored at 4°C for later use. The UV-Vis spectrophotometer proves that its absorption peak is at 529nm, corresponding to a particle diameter of 46nm ( figure 2 ).

[0046] 2. DNA modification of gold nanoparticles:

[0047] dna 1 (5′-SH-C6-ATCGTTTAGGATTTGGATGA-P-3′) and DNA 2 (3′-GCAAATCCTAAAC) configured as 10 -6 M solution, and then hybridize and incubate for one hour at 37°C on a mixing shaker. After the reaction is complete, add the hybridized DNA double strands into 1mL nano-gold solution. After stirring at room temperature for 24 hours, add ...

Embodiment 2

[0060] Fabrication of Photosensors

[0061] 1. Preparation of probes:

[0062] The synthesis of nano palladium: the freshly prepared Pd(NH of 100ml 0.01% (w / v) 3 ) 2 Cl 2 Mix the solution with 0.5g / L protective agent polyvinylpyrrolidone in a 10ml colorimetric tube, add the reducing agent polyethylene glycol under mechanical stirring, seal it, and put it in a constant temperature water bath at 60°C. As the reaction progresses, the color of the solution begins Change, stop heating when the solution turns black and does not change to obtain a nano-palladium mixed solution. The mixed solution was centrifuged twice for 15 min at 12000 rpm per minute, and the supernatant was skimmed off to obtain palladium nanoparticles. Re-disperse the nano-palladium in the nano-silver in the Tris-HCl solution containing 300mM sodium chloride and 50mM, and store it at 4°C for future use. Store it at 4°C for later use.

[0063] 2. Nano-palladium modification of DNA:

[0064] dna 1 (5′-SH-C6...

Embodiment 3

[0069] Fabrication of Photosensors

[0070] 1. Preparation of probes:

[0071] Synthesis of nano-silver: add 0.01g of bovine serum albumin into distilled water and stir until the bovine serum albumin is completely dissolved to form a clear and colorless solution, then add 5ml of 0.5M silver nitrate aqueous solution into it, place it on a magnetic stirrer, stir and mix evenly, add a certain amount Ammonia solution was made into silver ammonia solution, and then 0.2 g of ascorbic acid aqueous solution was quickly added to the silver ammonia solution, and stirring was continued for 10 minutes. The mixed solution was centrifuged twice for 15 min at 12000 rpm per minute, and the supernatant was skimmed off to obtain silver nanoparticles. The nano-silver was redispersed in a solution containing 300 mM sodium chloride and 50 mM Tris-HCl solution, and it was stored at 4° C. for future use.

[0072] 2. Nano-silver modification of DNA:

[0073] dna 1 (5′-SH-C6-ATCGTTTAGGATTTGGATGA-P...

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Abstract

The present invention discloses a photoelectric sensor for detection of kinase activity on the basis of local area surface plasma resonance. The biosensor is prepared as follows: a semiconductor material metal oxide is modified onto an indium tin oxide electrode (ITO), then Kemptide, PKA, and a probe are assembled onto the electrode layer by layer, since the probe contains noble metal nanoparticles and photosensitizer tris (bipyridine) ruthenium, under visible light irradiation, the photosensitizer tris (bipyridine) ruthenium captures more photons, local area surface plasma resonance effect of the noble metal nanoparticles occurs under photon excitation, so that more electrons jump onto a semiconductor metal oxide conduction band to produce photocurrent. The biosensor quantitative detection is based on that different concentrations of PKA cause different extents of phosphorylation of Kemptide, so that the amount of the probe connected to the modified electrode is also different, and photocurrent changes are caused. The method has high sensitivity and selectivity, enzyme inhibition experiments also show that the method achieves the purpose of efficient and sensitive detection of the kinase PKA activity.

Description

Technical field: [0001] The invention relates to the field of photoelectrochemical sensors, in particular to a photoelectric sensor for detecting kinase activity based on localized surface plasmon resonance. Background technique: [0002] Kinase (PKA)-regulated protein phosphorylation plays an important role in metabolic and cellular transduction pathways. Overexpression of protein kinases can cause a variety of diseases such as tumors, diabetes, Alzheimer's disease and so on. In the field of biochemistry, the detection of kinase activity and its inhibitors can elucidate the molecular mechanism of signal transduction. In the field of clinical medicine and drug loading, the early detection of abnormal kinase expression activity is also conducive to the prevention and treatment of diseases. [0003] Photoelectric biosensors based on the localized surface plasmon resonance effect utilize semiconductor metal oxides (such as: titanium oxide TiO 2 , Zinc Oxide (ZnO) properties t...

Claims

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Application Information

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IPC IPC(8): G01N21/552
Inventor 王宗花闫志勇赵凯夏建飞夏延致
Owner QINGDAO UNIV
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